Blood coagulation disorders are among the most prevalent clinical problems in the general population. Increased tendency for coagulation termed “Hypercoagulability” or “Thrombophilia” is a major cause of morbidity and mortality.
In the U.S. there are about 500,000 venous thrombosis events, with a conservative estimate of 100,000 deaths annually—greater than death occurrence related to AIDS, breast cancer and road accidents combined. In addition, 1.1 million myocardial infarctions, and more than 150,000 stroke deaths occur annually.
In cancer patients, thrombosis is the second leading cause of death, after the malignancy itself. Yet, therapy is given only after the occurrence of the thrombotic event.
In women, hypercoagulability is a major risk factor for pregnancy vascular complications including: thrombosis, severe preeclampsia, intra-uterine growth restriction and fetal death, and thrombosis following delivery or hormonal therapy. According to the recent literature, in developed countries the leading cause of death of women after delivery in pulmonary thrombo-embolism.
Another problem is the increased tendency for bleeding related to platelets, which is also common in the general population. About 25% of women with menorrhagia—increased menstrual bleeding—have such abnormality.
The major factor involved in pathogenesis of thrombosis and bleeding is the circulating blood platelets.
Properties of blood platelets may be measured by flow cytometry.
Flow cytometry (abbreviated: FCM) is a technique for counting and examining microscopic particles, such as cells, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders such as blood cancers.
A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of liquid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more fluorescent detectors. Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in light intensity at each detector (one for each fluorescent emission peak), it is then possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). This is because the light is scattered off of the internal components of the cell. See also: Tomer A [Tomer A 2004], [Tomer A et al., 1988], [Tomer A et al., 1989a] for further general introduction to FCM of blood.
Modern flow cytometers are able to analyze several thousand particles every second, in “real time,” and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers “high-throughput” (for a large number of cells) automated quantification of set parameters. For further general introduction to cell isolation and analysis, see Tomer A [Tomer A, 2002], [Tomer A et al., 1987].
Modern instruments usually have multiple lasers and fluorescence detectors. Increasing the number of lasers and detectors allows for multiple antibody labeling, and can more precisely identify a target population by their phenotypic markers [Tomer A, 2004]
The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed “gates.”
The following publications describe tests performed on blood platelets, involving cell cytometry.
U.S. Pat. No. 5,656,442 to SCRIPPS RESEARCH NST [US] describes methods for characterizing platelet aggregation defects. In one example a Cam variant of Glanzmann's thrombasthenia is characterized as having a ligand binding defect. In another, a patient with myelofibrosis is identified as having an activation defect. Analysis is by fluorescence-activated flow cytometry. The system includes an enclosure containing, in separate containers: (a) activation specific ligand (ASL) that binds with activated platelets: (b) an activation independent ligand (AIL) that forms a ligand-induced binding site (LIBS) on normal platelets, wherein said activation independent ligand includes a polypeptide listing a sequence selected from the group consisting of RGD, LGGAKQAGDV (SEQ ID NO:1), and KQAGDV (SEQ ID NO:2): (c) an anti-LIBS antibody; and (d) a platelet agonist.
WO 03028627 to BERG DAVID and BERG LOIS HILL [US] describes a method including tests for determining levels of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation by flow cytometry. Deviation from the normal values in any two of five assays is used to diagnose chronic fatigue syndrome, fibromyalgia, or other disease associated with activation of the coagulation response. No details are provided about measurement of the platelet activation.
US 2005214877 to PPD BIOMARKER DISCOVERY SCIENCES, LLC describes a method for measuring the amount of a platelet surface protein in a sample including platelets, including the steps of: (a) contacting the sample with a platelet stabilizing composition having an anticoagulant and at least one platelet activation inhibitor; (b) incubating the sample with a labeled compound having specific affinity for the platelet surface protein and a platelet stimulating factor; and (c) detecting labeled compound bound to platelets by cytometry (e.g. microvolume laser scanning cytometry), whereby the amount of the platelet surface protein may be measured.
US 2003194818 to HECHINGER MARK [US] describes immunoassay methods and apparatus which utilize flow cytometry, coated latex microspheres, and fluorochrome labeled antibodies, to simultaneously detect the presence and amount of one or more analytes in a sample. By combining FALS and fluorescence, beads of several different sizes, colors or shapes are used, each bead coated with a different analyte, for the simultaneous detection of one or more analytes and of cell components such as platelets in a sample.
US 2003032068 HECHINGER MARK [US] describes similar methods and apparatus, directed to platelet Ig positive control reagents and assays which provide for the setting of the fluorescence positive region for each patient. The platelet control is sized to fit between the platelets and red cells with the goal of making it ideal as a true biological control.
However, despite the clinical importance of platelet disorders there is yet an unmet need for platelet analysis systems and methods that would allow clinicians to easily diagnose various medical conditions related to platelets. Such conditions include both platelet functional abnormality, causing bleeding, and ongoing blood hypercoagulable activity, which may lead to vascular occlusion and thrombosis, and in pregnant women to placental vascular complications and fetal death. Currently used platelet analysis methods carry certain methodological and practical limitations, thus generally providing incomplete clinical information as is specified below.
Immune thrombocytopenia (IT) is a disorder characterized by antibody-mediated accelerated platelet destruction [George J N and Rizvi M A], [Tomer A et al. 1991]. Despite being a clinically important disorder its diagnosis is currently hampered by the lack of a feasible and reliable assay for routine clinical use. Thus, current diagnoses are generally based on clinical impressions deduced primarily by exclusion, see—[Neunert C et al.], [Provan D et al.], despite the patient presentations being sometimes complex.
Furthermore, suspected patients may be subjected to empiric therapies such as high dose corticosteroids that may carry significant side effects, or high dose intravenous immunoglobulins which is an expensive therapeutic option.
Methods to determine general anti-platelet or platelet bound antibody-similar to Coombs test for red-blood cells—have proven to be non-useful, since platelets unlike red cells express Fc-receptor and naturally bind circulating antibodies.
Current methods that may be used to determine autoimmune thrombocytopenia, such as an ELISA type assay (MAIPA), carry significant methodological and practical limitations, have limited specificity, are labor intensive (three-day work to obtain results) and require high expertise to obtain results [Chong B H, Keng T B], [Cines D B, Blanchette V S], [McMillan R, et al. 2003]. Thus they are not routinely available for diagnosis.
Further, these assays are not approved for the diagnosis of autoimmune thrombocytopenia.
For these reasons, no confirmatory laboratory assay is indicated or recommended by the American Society of Hematology [Neunert C et al.], [Provan D et al.] for the diagnosis of IT.
It is important to note that as indicated by Chong and Keng, however, the reason for not requiring a confirmatory test (as is required for example for the diagnosis of APS) is that there is not yet a reliable test with sufficiently high sensitivity and specificity. Furthermore, a diagnosis based on exclusion carries potential problems [Cines D B, Blanchette V S], [McMillan R, et al. 2003], thus a direct laboratory confirmation of the presence of circulating autoantibodies directed against platelet-specific receptors would be clinically helpful [Chong B H, Keng T B], [Cines D B, Blanchette V S], [McMillan R, et al. 2003]. The clinical effect of these antibodies is further highlighted by our previous studies [Tomer A, et al., 1989], [McMillan R. et al. 2004].
APS is an acquired hypercoagulable state affecting young and middle aged individuals. The syndrome is associated with arterial and venous thrombosis and in women, with recurrent fetal loss. The international diagnostic criteria require the occurrence of a clinical event, and the demonstration in the patient's blood of auto-antibodies reacting with natural phospholipids [Miyakis S, et al,]. Current laboratory assays for diagnosis are heterogeneous with methodological and practical limitations [Wong R C, Favaloro E J]. As stated in this reference “Despite numerous past and ongoing efforts, there remains significant variation in results from assays for the major antiphospholipid antibodies (aPL), namely anticardiolipin (aCL), anti-beta2 glycoprotein I (anti-beta2GPI), and lupus anticoagulant (LA)”, and “However, because of the paucity of good-quality published evidence, there is a heavy reliance on expert opinion, and thus the existing consensus guidelines for aPL testing and reporting are largely eminence based rather than evidence based”.
Another major problem is that the correlation of the laboratory findings with the clinical presentation is not entirely apparent. For example, according to several studies, about 15% to 17% of children with viral infection demonstrate APS false-positive test. In a recently reported study [de Groot], a world expert reported >30% misdiagnosis of APS samples sent by him to well established clinical laboratories. Regarding false positive and false negative results, see also [Merriman E et al.], [Aboud M et al.], [Pellegrino N M and Caccavo D], [Bizzaro N et al.], [Martorell J R et al.], [Koike T et al.], [Rusnak et al.], [Lakos G and Teodoescu M], [Moore G W et al.], [Pengo V et al.], [de Larrañaga G et al.], [Asherson et al.], [Zhu W F et al.], [Uthman I W et al.], [Bernard C et al.] for the methodological limitations, including false positive and false negative, etc. It is important to note that because of these methodological limitations of the routinely used laboratory tests, the international guidelines require the repetition of the assays 12 weeks apart when their outcome is positive [Miyakis S et al].
Heparin is the standard anti-coagulant therapy for the treatment and prevention of thrombosis. Heparin-induced thrombocytopenia and thrombosis (HIT) is an immune-mediated serious complication that may develop in patients sensitized to heparin. Approximately 5% of patients treated with full dose heparin develop HIT. About 50% of patients, who manifest HIT, develop thrombosis, half with severe morbidity and death. The diagnosis of HIT poses serious clinical dilemmas. At present, quick clinical decision is required to immediately discontinue heparin and start with an alternative anti-coagulant therapy, suitable for patients with HIT [Sheridan D, et al.], [Kelton J G, et al.], [Chong B H.], [Alving B.], [Aster R H.], [Thielmann M et al.]
Current methods based on the detection of antibodies against heparin-platelet-factor 4 complex such as the ELISA and the Gel-particle assay (e.g. PaGIA) have certain methodological and practical limitations.
Antibodies may be detected by these methods in up to 30% of patients treated with heparin, however, only 5% manifest clinical HIT [Sheridan D, et al.], [Kelton J G, et al.]. In addition, these assays have a range of >10% false-negative [Alving B.], [Arepally G, et al.,], [Hirsh J. et al.], [Visentin G P et al.], they detect only heparin-platelet factor 4 complex which is not formed in all patients. In addition, up to 80% false-positive results may occur in patients having autoimmune APS, i.e., the patient will carry life-long with unnecessary treatments on one hand and avoidance of required therapies on the other hand [Pauzner R, et al.].
The functional platelet aggregation assay (HIPA) is complex, requiring multiple normal donors (usually four), has a low sensitivity [Thielmann M et al.], [Chong B H et al.], [Favoloro E J et al.] and a low reproducibility. Furthermore, it involves platelet washing step, a manipulation known to cause platelet activation thus inevitably confounding the assay results.
The functional, radioactive serotonin-release assay (SRA) is considered the gold-standard, However, it is impractical and is not available out of limited research laboratories [Sheridan et al.], [Kelton J G et al.], [Alving B], [Arepally G et al.], [Visentin G P et al.], [Favoloro E J et al.]. Thus, to overcome these above mentioned limitations, we developed a practical, rapid, sensitive and specific functional flow cytometric method for the diagnosis of HIT. The functional method determines the capacity of patient's serum/plasma to induce platelet activation in presence of heparin—similar in concept to the gold-standard radioactive SRA.
Another method was described 15 years ago using flow cytometry [Tomer A, 1997]. However, because it has been found that this method requires high expertise, it is not available in regular clinical laboratories.
Defect in the hemostatic function of platelets leads to bleeding tendency-which not uncommon in the general population. Thus, testing of platelet function is an important clinical assessment.
Turbidometric aggregometry platelet function assessment method is a classic and most common method for testing platelet function, being used for approximately 50 years. It is based on stimulation of platelets in suspension and stirring with a magnet to form platelet aggregates, which allow more light transmission compared to full suspensions. A more modern instrument—though not very common—is the PFA100 of Siemens Co which imitates this reaction with a difference in the method of reading. The PFA-100 aspirates a blood specimen into disposable test cartridges through a microscopic aperture cut into a biologically active membrane at the end of a capillary. The membrane of the cartridges is coated with collagen and adenosine diphosphate (ADP) or collagen and epinephrine, inducing a platelet plug to form which closes the aperture.
The commonly employed methods use a relatively high-dose stimulant to achieve an end-point result, thus not being capable of testing the three phases of the platelet activation process leading to final aggregation, as the method described here does, which is important in diagnosis of platelet dysfunction.
In addition, the use of high-dose stimulants abrogates the possibility of detecting mild to moderate dysfunction such as occurs in platelet storage-pool disease [B S Coller and D L French], [Shattil S J et al.], [Fitzgerald R, Pirmohamed M.]. These methods also incapable of detecting some platelet functional disorder such as Scott syndrome and other ones [B S Coller and D L French], [Shattil S J et al].
Blood platelets play a pivotal role in normal hemostasis. Paramount to their function are membrane glycoproteins (GPs) that specify the critical ligand interactions involved in platelet adhesion and aggregation, necessary for normal hemostasis.
Congenital platelet dysfunctions are heritable bleeding disorders that may result from platelet glycoprotein-receptor abnormalities. As a consequence, these disorders are associated with excessive bleeding, especially from skin and mucosa. Bernard-Soulier syndrome and Glanzmann thrombasthenia are the major congenital disorders of platelet-receptor defects [B S Coller and D L French], [Shattil S J et al.], [Fitzgerald R, Pirmohamed M.].
Bernard-Soulier syndrome results from a defect in the GP Ib-IX (CD42) complex, which functions as a binding site for the von Willebrand factor (vWF), which in turn mediates platelet attachment to components of subendothelium, exposed by damage to the vessel wall [B S Coller and D L French], [Nurden A, Nurden P.], [Harold R Robert and Alice D Ma], [Shattil S J et al.].
This syndrome is also associated with thrombocytopenia. Therefore, it is frequently confused with Immune Thrombocytopenia (IT), as occurs for example with the index patient shown in FIGS. 13-14, who was planned for an unnecessary surgical procedure—splenectomy. It is important to note here that some platelet disorders as this require sometimes more than one test to achieve correct diagnosis-such as ruling out IT in the present case. Thus, the proposed Platelet Analysis System here is merely a one system for evaluation of platelet disorders.
Glanzmann thrombasthenia results from a defect in the major platelet functional receptor GPII/IIIa (CD41a), necessary for fibrinogen-mediated platelet aggregation [B S Coller and D L French], [Shattil S J et al.], [Fitzgerald R, Pirmohamed M.].
Aspirin inhibits the arachidonic acid pathway enzyme cyclooxygenase I, COX-I), which is required for the formation of the platelet prostaglandin stimulant, Thromboxane A2, in a coagulation process.
Thienopyridine agents specifically and irreversibly inhibit the P2Y12 sub-type of ADP receptor, which is important in platelet activation and aggregation [Shattil S J et al.].
Current clinical guidelines recommend a chronic treatment with Platelet-inhibitory agents for all patients with coronary-artery disease (CAD), peripheral vascular disease (PVD), cerebro-vascular disease (CVD) that includes brain circulation limitations, patients with transient-ischemic-attack (TIA), or stroke; retinal vascular thrombosis in the eye, vascular angioplasty (such as coronary artery dilation by catheter—with or without stenting), and other categories of patients with risk of vascular occlusion and thrombosis.
Nevertheless, many patients with recurrent thrombosis have been found not to have adequate response to the inhibitory effect of these agents, a syndrome termed “Aspirin resistance” [Fitzgerald R, Pirmohamed M.], or “Clopidogrel resistance” [Qureshi Z, Hobson A R.].
There is need for a practical system for diagnosis that includes instrumentation and diagnostic kits. The proposed system should allow the performance of feasible and highly informative laboratory assays. The assays should be highly reliable and capable of providing useful medical information for the most common platelet-related disorders.
A special aim in the design of the system is to provide highly needed tests for the determination of circulating platelet activation markers as indicators of ongoing, in vivo, prothrombotic activity. These tests are not available by the commonly used platelet analysis methods in the clinical coagulation laboratories.
All proposed assays are optimized, simplified, refined and adjusted for daily routine use in clinical laboratories.